Skeletal muscle contraction is initiated by a proposed physical interaction between two enormous ion channel complexes, the dihydropyridine receptor (DHPR) in the sarcolemma and the ryanodine receptor Ca2+ channel (RyR1) in the sarcoplasmic reticulum (SR). The DHPR ?1S subunit is essential to couple this protein to RyR1, but this interaction cannot occur without the DHPR ?1a subunit, which plays a pivotal but poorly understood role in excitation- contraction (EC) coupling. While all three of these proteins are absolutely required for skeletal muscle function, how they fit together to produce the EC-coupling signal in the triad junction of higher vertebrate remains as one of the most fundamental unanswered question of muscle biology. The long-term goal of this proposal is to identify these interactions and define their interrelationship under normal and myopathic conditions. Consequently, here we propose a systematic structure/function characterization of the DHPR/RyR1 complex in its native skeletal muscle environment using an innovative multidisciplinary approach. In Aim-1 we propose a new model of association between DHPR complexes. Here we will test the role of leucine zipper motifs of ?1a and ?1S subunit in both interlinking adjacent DHPR particles and in EC- coupling signaling. These studies will use a multi-disciplinary approach involving site-directed mutagenesis, Ca2+ imaging, whole-cell patch clamp and freeze-fracture analyses in mouse cultured myotubes. In Aim-2 we will use an innovative FRET-based approach to map the position(s) of critical domains of the DHPR complex relative to each other within intact myotubes. We will also determine how the structure of the DHPR complex is affected by the disruption of the critical leucine zippers as well as how it adjusts during EC-coupling under normal and pathophysiological conditions (malignant hyperthermia syndrome). In Aim-3 we will use our FRET-based assay to determine the orientation of the DHPR complex in relationship to key functional domains of RyR1 implicated in EC coupling. These studies will both identify sites of physical interaction between the two channels and will help to determine the relative orientation of the DHPR and RyR1, directly testing our working model. Successful completion of this proposal should provide with a detailed structural map of critical inter-molecular interactions required for skeletal-type EC-coupling, therefore, provide with essential information to understand physical coupling between these channels in health and disease.